Our commitment to studying the role of calcium effector pathways in ovarian differentiation will be continued by focusing on the regulatory actions of the calcium-phospholipid-diacylglycerol (PKC) second-messenger system in a well defined and established in vitro model of swine granulosa cells that retain responsivity to a variety of hormonal agonists. We will particularly investigate the manner in which FSH and angiotensin II (presumptively acting respectively through distinct cyclic AMP and PKC intracellular signaling pathways) alter intracellular calcium ion concentrations in single porcine granulosa cells (Time-resolved fluorescence video-microscopy); how hormonal agonists trigger increased secretory activity by single ovarian cells (reverse hemolytic plaque assay of relaxin secretion by pig luteal cells); employ a microperifusion system to test the temporal concordance between intracellular calcium-ion concentration changes and secretory events; probe the causal association between increased intracellular calcium-ion concentrations and resultant secretion using experimentally induced calcium transients in permeabilized cells; and begin to dissect the important interactions between the calcium effector signaling pathway and the cyclic AMP second messenger pathway on sterol-metabolizing enzymes. These emphases reflects work carried out under my parallel RCDA, in which I have has occasion to learn appropriate single-cell and reproductive biology techniques that can now be applied to this R01 renewal. The significance of the calcium second-messenger system in the ovary will therefore be explored at the single cell level (Specific Aim I), in relation to calcium's causal relevance to hormone secretion (Specific Aim II), and in relation to the interactions between the PKC and classical cyclic AMP effector pathways (interactions to be studied at the level of steroidogenesis). Overall, we anticipate that these approaches will yield significant and interesting new information regarding ovarian cellular physiology, which will enhance our physiological understanding or reproductive biology and fertility regulation in higher mammals.